PhD Programme in Experimental Medicine and Therapy - Dottorato in Medicina e Terapia Sperimentale

Regulatory role of limbic neuropeptide Y Y₁ receptor on body weight and anxiety: modulation by maternal care in conditional knockout mice

 

 

XXII cycle PhD student: Ilaria Bertocchi

Tutor:

 

NPY is widely distributed in the CNS, where it is involved in the regulation of anxiety, stress reactions, energy balance, circadian rhythms and cognition ^{1, 2}. Preclinical and clinical studies suggest that NPY plays an important role in the response to stress and in psychiatric disorders ³. Haplotype-driven expression of NPY in humans predicts brain responses to emotional and stress challenges and inversely correlates with trait anxiety ⁴. Intracerebroventricular injection of NPY reduces both anxiety- and stress-related behavior in several animal models, an action that is primarily mediated by Npy1r in the amygdala, hippocampus, locus coeruleus and septum ^{1, 5}. The implications of endogenous NPY acting via Npy1r in the control of emotionality, mood and stress reactions have been probed with Npy1r-selective antagonists and antisense oligonucleotides ^6-9. NPY exerts its anxiolytic-like effect in the brain via interactions with hypothalamic-pituitary-adrenocortical (HPA) axis and corticosteroids. Indeed, a functional antagonism between NPY and CRH has been demonstrated in various nuclei along the stress/anxiety circuits ^{1, 5}.

In addition to its crucial role in emotional behavior, NPY stimulates feeding behavior through activation of  Npy1r,  which is expressed in the hypothalamic sites involved in the daily regulation of ingesting behavior and energy balance, i.e. the arcuate, paraventricular, ventromedial and dorsomedial nuclei and in the lateral hypothalamic area ^{1, 10}. Moreover, peripheral sympathetic NPY mediates the weight gain associated with stress and a hyper caloric diet through activation of Npy2r in white adipose tissue ¹¹.

So far, studies on mutated mice lacking the Npy1r have shown that phenotypes either exhibit minimal changes or they are conflicting when compared to pharmacological studies. Npy1r germinal knockout mice show an inconsistent anxiety-like phenotype, which is dependent on task and circadian rhythm ¹². Moreover, despite the potent orexigenic action of NPY, mutated mice lacking the Npy1r do not display any major abnormalities in their feeding behavior or body weight. Paradoxically, these mice develop late onset obesity, with a significant increase in body fat mass, and hyperinsulinemia, particularly in the females ^13-15.

To circumvent some of the problems inherent to classical knockouts, such as possible compensatory effects during development, and to unmask the functional role of Npy1r only in those areas most directly modulating anxiety and stress-response, we restricted the inactivation of the Npy1r gene in excitatory neurons of the forebrain by using the dox controlled Cre expression system ^{16, 17}.

In this genetic paradigm, region-specific and postnatal inactivation of the loxP site tagged Npy1r gene can be achieved in juvenile mice by transgenic tTA activated Cre expression. Therefore, we generated two Npy1r^loxP/loxP sublines: one containing the Tg^{α-CamKII-tTA} transgene  expressing the tTA in excitatory neurons of the forebrain under the control of the α-CamKII promoter¹⁸, and a second one bearing the transgene containing the tTA-responsive promoter for Cre expression (Tg^LC1 mice¹⁹). Region-specific and postnatal inactivation of the Npy1r gene can now be achieved in offspring of Npy1r^loxP/loxP/Tg^{α-CamKII-tTA} and Npy1r^loxP/loxP/Tg^LC1 intercrossed mice which received dox (50mg/l) during the mating to suppress Cre expression during embryonic development in their offspring. Offspring were genotyped and Npy1r^loxP/loxP/Tg^{α-CamKII-tTA/LC1}, Npy1r^loxP/loxP, Npy1r^loxP/loxP/Tg^LC1 and Npy1r^loxP/loxP/Tg^{α-CamKII-tTA} genotypes were found at the expected ratio.

Given that maternal behavior induces long lasting consequences on offspring’s emotional behavior, physiology and gene expression^{20, 21}, we postulated that variation in maternal care of foster dams might lead to long-term developmental effects in control mice that could be determinant to unmask the phenotype of Npy1r^fb-/- mice. Therefore, the expression of Cre was induced by fostering the litters from P0 using dox-naïve C57BL6/J and FVB/J mothers to include differences in maternal care in our phenotypic analysis. The removal of dox activates the expression tTA dependent of CRE and inactivates the Npy1r^loxP/loxP allele by exon 2 and 3 removal within the first three weeks in development of Npy1r^loxP/loxP/Tg^{α-CamKII-tTA/LC1} (Npy1r^fb-/-)mice.

In Tg^{α-CamKII-tTA/LC1} positive offspring Cre specific immunosignal increased over time in the cortex, hippocampus and amygdaloid nuclei reflecting postnatal increase in tTA from the transgenic α-CaMKII promoter. The Cre expression pattern was confirmed by the lacZ Cre reporter mouse line Gt(ROSA)26Sor ²², which showed similar regional restricted distribution of Cre immunoreactivity when Cre expression was suppressed in Gt(ROSA)26Sor/Tg^{α-CamKII-tTA/LC1} embryos, and no significant differences were observed in mice raised by either FVB/J or C57BL/6J dox free foster mothers.

Conditional inactivation of Npy1r was verified by immunohistochemistry of Npy1r peptide and by in situ hybridization of Npy1r mRNA. Npy1r^loxP/loxP control mice fostered to FVB/J mothers showed the normal widespread expression pattern of Npy1r mRNA and Npy1r immunostaining in the CNS, as previously described²³. In Npy1r^fb-/- conditional mutants reared by FVB/J mothers, Cre recombination took place in principal neurons of the anterior forebrain, including the hippocampal CA1 and CA3 pyramidal and dentate gyrus (DG) granule cell layers, cerebral cortex and amygdala, detected by a significant reduction of Npy1r mRNA expression in these brain regions. Npy1r protein and mRNA expression were unaffected in the hypothalamus of Npy1r^fb-/- mice. In contrast, in Npy1r^loxP/loxP mice reared by C57BL/6J mothers the signal for Npy1r expression was to low in in situ hybridization and immunoblots and could not be used to verify the and quantify mRNA.

Since the difference in Npy1r expression was drastically different in FVB/J and C57BL/6J fostered offspring we characterized individual differences in maternal behavior of FVB/J and C57BL/6J foster dams through direct observation of mother–pup interactions. The overall time spent in nursing behavior by FVB/J and C57BL/6J dams was not significantly different. However, FVB/J dams spent more time in crouching over the pups in an active form of nursing known as arched-back nursing (ABN^{24, 25}) as compared to C57BL/6J dams. Moreover, FVB/J foster mothers spent more time in eating/drinking and in resting outside the nest but showed lower activity and self-grooming than C57BL/6J dams. In addition, we found a strain difference in the dams’ behavior when tested in the open field (OF) and elevated plus maze (EPM) after litters’ weaning. In both tests, C57BL/6J females displayed higher anxiety-like behavior compared to FVB/J females. This variation in trait anxiety might be related to the reported strain difference in maternal behavior toward fostered pups.

Consistent with these variations in maternal care, litters fostered to C57BL/6J dams showed a lower body weight growth curve starting from P6; also after weaning and as adults, all mice reared by C57BL/6J foster mothers showed a reduced body weight when compared to FVB/J fostered mice, independent from the genotype. In contrast, conditional mutant mice reared by FVB/J foster mothers showed a lower body-weight growth curve compared to their respective controls, starting at approximately P35, when Cre-mediated inactivation of Npy1r is fully achieved. This difference in body weight increased with age and persisted throughout the 7 months period of monitoring, when Npy1r^fb-/- conditional mutant mice weighted approximately 20% less than their control littermates (Npy1r^loxP/loxP: 38 ± 3.0 g ; Npy1r^fb-/-: 30 ± 1.2 g, n =6; t: 2,728; P<0.05 by student t test). Thus the persistence of the Npy1r induced expression in the limbic system of adult mice seems to be important for the maintenance of a phenotype which is induced by a high maternal care of FVB/J foster mothers.

Decrease in body weight of Npy1r^fb-/- adult mice was associated with decreased leptin plasma levels and hyperglycemia in fed mice. Thus provide the first evidence for the involvement of limbic Npy1r in the regulation of energy homeostasis. In Npy1r^loxP/loxP and Npy1r^fb-/- mice reared by C57BL/6J foster mothers, the leptin and glucose values were not significantly different compared to Npy1r^fb-/- mice reared by FVB/J foster mothers, which is consistent with the low expression of Npy1r in these mice.

The exact role of forebrain Npy1r in the control of energy balance remains unknown. Central administration of Npy1r agonists stimulates food intake in rodents, an effect that is associated with stimulation of Npy1r in the hypothalamus¹. Here we showed that forebrain-specific Npy1r deleted mice show no significant changes in basal food intake or fasting-induced refeeding. Notably, while food consumption was similar among groups under normal conditions, we observed that during the first 24 hours following the 30 hours-fasting, mice adopted by C57BL/6J mothers showed a statistically significant increase in food intake as compared to pups reared by FVB/J, suggestive of an increased response to acute stress by the adult offspring of C57BL/6J foster mothers. However, weight gain after re-feeding was similar to that observed in mice fostered to FVB/J dams.

Furthermore, spontaneous locomotor activity in home cages was similar in control and mutant mice. These data therefore suggest that the observed decrease of body weight in Npy1r^{fb -/-} mice cannot be attributed to differences in feeding behavior or activity levels but it might possibly depend on changes in thermogenesis or basal energy expenditure rate. Previous studies have shown that inactivation of  Npy or Npy2r in the hypothalamus of adult mice induces small or indiscernible effects on appetite and transiently affects body weight, consistent with adaptation to maintain homeostasis^{26, 27}. It is interesting to note that forebrain specific deletion of Npy1r induces a permanent decrease in body weight, indicating that the loss of Npy1r in the limbic system cannot apparently be compensated by such adaptive mechanisms. Therefore the function of Npy1r in the hypothalamic sites involved in the daily regulation of ingesting behavior and energy balance ^{1, 15, 28} has to be reevaluated. Given that NPY/Npy1r signaling antagonizes CRH also in PVN²⁹, it is possible to speculate that selective disruption of limbic Npy1r might affect body weight gain via HPA axis.

This might find support in our findings that Npy1r^fb-/- mutant mice also showed decreased density of CRH-immunoreactive fibers. Plasma corticosterone concentrations in all C57BL/6J fostered and FVB/J fostered Npy1r^fb-/- mice were weakly, although not significantly, increased, when compared to FVB/J fostered Npy1r^loxP/loxP control mice with strongest Npy1r expression. Nevertheless, the conditional mutants reared by FVB/J mothers displayed a significant decrease of  CRH immunoreactive fibers in the central amygdala (CeA) when compared to their controls littermates. Similarly, the Npy1r^loxP/loxP mice raised by C57BL/6J dams showed a marked reduction in CRH immunoreactivity in the CeA when compared with Npy1r^loxP/loxP mice raised by FVB/J mothers.

A functional antagonism between NPY and CRH has been demonstrated in various nuclei along the stress/anxiety circuits suggesting that NPY may act as an endogenous agent to buffer against the stressor-induced release of CRH^{1, 30, 31}. Moreover, glucocorticoids stimulate the NPY system via an inhibition of CRH^{32, 33}. Reduced CRH expression may involve altered glucocorticoid negative feedback and/or increased CRH release concomitant with a failure of acute stress-induced facilitation of CRH production³⁴.

Selective disruption of NPY/Npy1r signaling pathways in the forebrain significantly increased anxiety-related behavior. Anxious behavior of Npy1r^fb-/- conditional knockout mice was observed in two different behavioral tests, the EPM and the OF. In the EPM, Npy1r^fb-/- mutants showed a lower frequency of entries and spent significantly less time in the open arms. The anxiogenic effect of region-specific inactivation of Npy1r was confirmed in the OF test. Although control and mutant mice spent a similar percentage of time in the center of the arena, Npy1r^fb-/- conditional knockout mice displayed significantly higher immobility in it, suggestive of anxiety-induced “freezing” behavior. In both tests, behavioral analysis of mice reared by C57BL/6J foster mothers revealed no differences between Npy1r^fb-/- and Npy1r^loxP/loxP mice that, in turn, showed lower frequency of entries and lower time in the open arms in the EPM test, and higher immobility in the center of the arena and lower total distance travelled in the OF when compared with Npy1r^loxP/loxP mice raised by FVB/J foster dams. Such a difference in anxiety responses between control and conditional mutant mice reared by FVB/J foster mothers was not primarily due to impairment of locomotor activity since spontaneous locomotor activity in the home cage did not differ.

In summary, we established a new genetic tool to study Npy1r function in the forebrain of adult mice. A detailed behavioral and molecular analysis revealed that conditional inactivation of Npy1r in neuronal circuitries in the CA1, CA3 regions and BLA mediate anxiety-related behavior. We provide the first genetic evidence that limbic Npy1r is required for regulation of body weight and it cannot apparently be compensated by adaptation to maintain homeostasis. Finally, our data further indicate that the Npy1r upregulation is a target of maternal care and that the limbic NPY/Npy1r neuronal pathway is responsible for the persistent effects of maternal care on anxiety-related behavior and energy homeostasis.

The mechanisms whereby maternal care is able to influence Npy1r mRNA expression and function remain to be determined. We suggest two main working hypotheses.

Recent studies address the possibility that environmental events might alter chromatin structure through effects on DNA methylation and thus affect GR gene expression³⁵. We are currently investigating if maternal care can induce stable changes on limbic Npy1r by epigenetic mechanisms. On the other hand, given that glucocorticoids stimulate the NPY system via an inhibition of CRH^{32, 33}, it is possible that an altered glucocorticoid negative feedback could induce a decrease of NPY-Npy1r function.

It is important to note, however, that our experiments do not exclude the possibility that milk composition or some other aspect of nursing may also play an important role in pups’ development under normal conditions. In fact, Levine’s work has demonstrated that milk provided by the mother plays an important role in inhibiting the ability of the adrenal gland to secrete corticosterone in response to adrenocorticotropic hormone stimulation³⁶.

 

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